Project Summary: To date the mechanisms that specify individual neuronal fates during regeneration are poorly understood. Determining how regenerative specification programs promote successful regeneration is an essential step to improve the design of regenerative therapies. However, to date we lack sufficient examples of regenerative neuronal patterning, which hampers our ability to better determine how the proper neuronal fates are generated during regeneration. We do know that regeneration doesn't recapitulate developmental patterning, which suggests that changes to fate known specification programs are necessary for regenerative neurogenesis. Because regeneration doesn't recapitulate development, being able to compare how changes to the developmental patterning programs result in regenerative success will provide key insights that will improve our understanding of how to promote regeneration. Thus, there is a critical need to identify regenerative neuronal patterning mechanisms in animals suited for comparing the developmental and regenerative programs that generate identical cell types. The PI's long-term goal is to understand how regenerative specific patterning programs promote successful regeneration. To advance this goal the PI developed the highly regenerative and excellent developmental system the sea anemone Nematostella vectensis as a model to investigate developmental and regenerative neurogenesis. The PI has identified a neurogenic transcription factor (NvashA) that is differentially deployed during regeneration and development, indicating that its regenerative function is different than its developmental role. Similarly, we also identified a class of neurons described by the NvLWamide::mcherry reporter. During development these neurons require NvashA for proper specification. Three of the five subtypes of NvLWamide neurons show differences between their developmental and regenerative formation. This work will use a series of conditional alleles and in vivo imaging to functionally determine if some of the regeneration specific differences in NvLWamide fate specification are explained by changes in their requirement for NvashA. To determine how NvashA functions during regeneration the regenerative targets of NvashA will be identified using an RNAseq approach. Lastly, to gain insights about how new NvLWamide neurons are patterned during regeneration, and to gain insights about how remnant neurons reintegrate during neuronal regeneration the transcriptomes of regenerating NvLWamide neurons will be determined at multiple time points throughout regeneration, and NvLWamide genes that change over time will be mapped to newly forming or remnant neurons. This work will demonstrate that developmental patterning genes play distinct roles during regeneration, and that neuronal cell types require new specification programs to regenerate. The foundation of data generated in this proposal will allow the PI to make a sustained impact in the field of regenerative neurogenesis by elucidating the mechanisms that promote regenerative neuronal patterning in future studies spawned from the efforts described here.